Reproduction in women depends upon the dynamic interaction of several compartments of the female reproductive system. The hypothalamic-pituitary unit orchestrates a series of events affecting the ovaries and the uterine-endometrial compartment which leads to the production of the ovum, ovulation, and ultimately appropriate conditions for fertilization. Specifically, hypothalamic hormones enhance the release of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH). In the ovary, gonadotropins enhance the development of follicles which, in turn, secrete steroids (estradiol, progesterone) and peptides (inhibin, activin). Estradiol and inhibin levels progressively increase during the follicular phase of the menstrual cycle until ovulation. Afterwards, the follicular unit forms the corpus luteum which produces progesterone. Ovarian hormones, in turn, regulate the secretion of gonadotropins by establishing a classical long-loop negative feedback mechanism. The elucidation of these control mechanisms has provided opportunities for the development of effective strategies to control fertility, including both the enhancement of fertility and contraception. For recent reviews of FSH action see xe2x80x9cFSH Action and Intraovarian Regulationxe2x80x9d, B. C. J. M. Fauser, editor, Parthenon Publishing Group, 1997 and Hsueh, A. J., Bicsak, T., Jia, X.-C., Dahl, K. D., Fauser, B. C. J. M., Galway, A. B., Czwkala, N., Pavlou, S., Pakoff, H., Keene, J., Boime, I, xe2x80x9cGranulosa Cells as Hormone Targets: The role of Biologically Active Follicle-Stimulating Hormone in Reproductionxe2x80x9d Rec. Prog. Horm. Res., 1989, 45, 209-277.
Current hormonal contraception methods are steroidal and take advantage of long-loop feedback inhibition of gonadotropin secretion, as well as effecting peripheral mechanisms such as sperm migration and fertilization. An alternative strategy for hormonal contraception would be the development of specific antagonists of the receptor for FSH. Such antagonists would disrupt the actions of FSH on follicular development, thus producing the desired contraceptive effect. The utility of this strategy is supported by mechanism of infertility of women with resistant ovary syndrome. The infertility experienced by these women is the result of non-functional FSH receptors (K. Aittomaki, J. L. D. Lucena, P. Pakarinen, P. Sistonen, J. Tapanainnen, J. Gromoll, R. Kaskikari, E.-M. Sankila, H. Lehvaslaiho, A. R. Engel, E. Nieschlag, I. Huhtaniemi, A. de la Chapelle xe2x80x9cMutation in the Follicle-Stimulating Hormone Receptor Gene Causes Hereditary Hypergonadotropic Ovarian Failurexe2x80x9d Cell, 1995, 82, 959-968). This approach to contraception also appears applicable to men, since idiopathic male infertility seems related to a reduction in FSH binding sites. Moreover, men with selective FSH deficiency are oligo- or azoospermic with normal testosterone levels and present normal virilization. Therefore, orally active FSH antagonists may provide a versatile method of contraception.
Suramin Sodium, is an anticancer agent with a wide variety of activities. Recently suramin was shown to inhibit FSH binding to its receptor (Daugherty, R. L.; Cockett, A. T. K.; Schoen, S. R. and Sluss, P. M. xe2x80x9cSuramin inhibits gonadotropon action in rat testis: implications for treatment of advanced prostate cancerxe2x80x9d J. Urol. 1992, 147, 727-732). This activity causes, at least in part, the decrease in testosterone production seen in rats and humans that were administered suramin (Danesi, R.; La Rocca, R. V.; Cooper, M. R.; Ricciardi, M. P.; Pellegrini, A.; Soldani, P.; Kragel, P. J.; Paparelli, A.; Del Tacca, M.; Myers, C. E, xe2x80x9cClinical and experimental evidence of inhibition of testosterone production by suramin.xe2x80x9d J. Clin. Endocrinol. Metab. 1996, 81, 2238-2246). Suramin is the only non-peptidic small molecule that has been reported to be an FSH receptor binding antagonist. 
Compounds of formula A are described in the literature as follows: 
Y. Yamashita [Yuki Gosei Kagaku Kyokai Shi (1970), 28(10), 1025-31] disclosed compounds of formula A (U, V, X, Y, Z=H) (U, V, Y, Z=H, X=p-NO2) (U, V, Y, Z=H, X=o-NO2) (U, V, Y, Z=H, X=p-OMe) (U, V, Y, Z=H, X=o-OMe) (U, V, Y, Z=H, X=p-Cl) (U, V, Y, Z=H, X=m-Cl) as fluorescent whitening agents.
Y. Yamashita [Yuki Gosei Kagaku Kyokai Shi (1971), 29(5), 519-25] disclosed a compound of formula A (U, V, Y, Z=H, X=o-Cl) as fluorescent whitening agents.
J. H. P. Tyman [J. Soc. Dyers Colour. (1965), 81, 102-104] disclosed a compound of formula A (U, V, Y, Z=H, X=p-CH3).
Yura et al. [Kogyo Kagaku Zasshi (1955) 58, 664-665; CA 1956; 11675] disclosed compounds of formula A (U, V, Y, Z=H, X=p-NHCH3) (U, V, Y, Z=H, X=p-NHCOCH3) (U, V, Y, Z=H, X=p-NHCOPh) (U, V, Y, Z=H, X=p-NHCH2OH).
D. W. Hein and E. S. Pierce [J. Am. Chem. Soc. 1954 (76) 2725-2730] disclosed compounds of formula A (U, V, Y, Z=H, X=o-OEt) (U, V, Y, Z=H, X=o-OPh) (U, V, Z=H, X=o-OMe, Y=p-OMe) (X, Y, Z=H, U=5-Cl, V=5xe2x80x2-Cl) (Y, Z=H, X=p-OMe,U=5-Cl, V=5xe2x80x2-Cl) (Y, Z=H, X=o-OEt, U=5-Cl, V=5xe2x80x2-Cl) (Y, Z=H, X=o-OPh, U=5-Cl, V=5xe2x80x2-Cl) (Z=H, X=o-OMe, Y=p-OMe, U=5-Cl, V=5xe2x80x2-Cl) (Y, Z=H, X=o-OEt, U=6-Cl, V=6xe2x80x2-Cl) (Z=H, X=o-OMe, Y=p-OMe, U=6-Cl, V=6xe2x80x2-Cl).
R. D. Haugwitz, L. Zalkow, E. Gruszecka-Kowalik and E Burgess (WO 9625399) disclosed compounds of formula A (U, V, Y, Z=H, X=o-OH) (U, V Z=H, X=p-NH2, Y=o-SO3H) (U, V, Z=H, X=p-NO2, Y=o-SO3H) (U, V=H, X=o-CO2H, Y=o-OH, Z=m-OH) (U, V Z=H, X=o-OH, Y=m-CH2SCH2CH2CO0H) for treatment of viral infections.
H. J. Roberts (U.S. Pat. No. 3453262) disclosed the compound of formula A (U, V, Z=H, X=p-OMe, Y=p-OMe) as a fluorescent brightening agent.
I. V. Aleksandrov and G. E. Samoliva [Deposited Publ. (1972), Issue VINITI 4341-72; CAN 85:32589] disclosed the compound of formula A (U, V, Y, Z=H, X=p-CN).
B. D. Gummow, G. A. F. Roberts [Makromol. Chem. (1986), 187(4), 995-1004] disclosed the compound of formula A (U, V, Y, Z=H, X=p-NH2).
A compound of formula A (U, V, Y, Z=H, X=m-NH2) was disclosed in DE 250342 (CA 6421-83-6; Beilstein 3526749).
F. Fleck [ Swiss 318,441 (1957)] disclosed compounds of formula A (U, V, Y, Z=H, X=o-OCH2CHxe2x95x90CHCH3) (U, V, Y, Z=H, X=m-OCH2CHxe2x95x90CHCH3) (U, V, Y, Z=H, X=o-OCH2CH=CH2) (U, V, Y, Z=H, X=p-OCH2CHxe2x95x90CH2) (U, V, Y, Z=H, X=m-OCH2CHxe2x95x90CH2).
R. Fleischhauer, F. Aldebert [Ger. 1,011,889 (1957)] disclosed compounds of formula A (U, V, Y, Z=H, X=p-OCH2C(CH3)=CH2) (U, V, Y, Z=H, X=m-OCH2C(CH3)xe2x95x90CH2).
M. Pestemer, A. Berger, A Wagner [Fachorgan Testilveredlung (1964), 19(6) 420-5] disclosed the compound of formula A (U, V, Z=H, X=o-OMe, Y=p-Me).
R. S. Long, A. K. Kantor [U.S. Pat. No. 2,848,484 (1958)] disclosed the compound of formula A (Z=H, X=o-OMe, Y=p-OMe, U=5-OMe, V=5xe2x80x2-OMe).
A. Mitrowsky, O. Bayer [Ger. 937,822 (1956)] disclosed the compound of formula A (U, V=H, X=o-OMe, Y=m-Me, Z=p-Me).
R. S. Long, A. K. Kantor [U.S. Pat. No. 2,841,613 (1958)] disclosed the compound of formula A (Z=H, X=o-OMe, Y=p-OMe, U=5-OMe, V=5xe2x80x2-Cl).
K. W. Eder [U.S. Pat. No. 2,806,054 (1957)] disclosed compounds of formula A (U, V, Y, Z=H, X=p-OCHCH2OAc) (U, V, Y, Z=H, X=p-OCH2CH2OCOPh).
U.S. Pat. No. 2,567,796 was disclosed compounds of Formula B. 
A compound of formula C is disclosed by I. G. Macara, S. Kuo, and L. C. Cantley in J. Biol. Chem. (1983), 258(3), 1785-92. 
A compound of formula D is disclosed by A. Froehlich, in Ger. Offen., 47 pp. Addn. To Ger. Offen 1,917,813 (CA 73;50737y), DE 2161818 (1972). 
Compounds of formula E are disclosed by J. M. Farrar in GB2203426 (1988), where RY and RZ are independently substituted alkylamino, alkoxy and alkylthio groups]. 
None of the aforementioned compounds are disclosed to be follicle stimulating hormone (FSH) antagonists or contraceptive agents. Addtionally, none of the aforementioned compounds contain the appropriate substitutions on the pendant benzoyl ring or sulfonate-containing ring required for good activity as FSH antagonists or contraceptive agents contained on the compounds of the present invention.
The compounds useful in this invention have the general formula (I) 
wherein
Q is hydrogen or xe2x80x94SO2R1;
X is a bond, O, S(O)n, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CH2CH2xe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, or xe2x80x94CH2S(O)nCH2xe2x80x94;
R1 is OH, NH2, C1 to C6 alkoxy, C1 to C3 perfluoroalkoxy; 
R2 and R4 are each, independently, hydrogen, OR6, xe2x80x94S(O)mR6, xe2x80x94NHR6, xe2x80x94N(R6)2, or xe2x80x94CH2SO2CH3;
R3 and R5 are each, independently, hydrogen, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94SO2R9, or xe2x80x94CH2R9;
R6 is hydrogen, C1 to C6 alkyl, C3 to C6 alkenyl, xe2x80x94CH2CH2Z, xe2x80x94CH2COR7, xe2x80x94CH2CHxe2x95x90CHCOR7, 
Y1 and Y3 are each, independently, N, or CH;
Y2 and Y4 are each independently, O, S, or NR13;
R7 is xe2x80x94OR8, xe2x80x94NHR8, xe2x80x94N(R8)2, or xe2x80x94NHCH2CH2OR8;
Z is xe2x80x94OR8, xe2x80x94OCH2CH2OR8, xe2x80x94N(R8)2, or 
R8 is hydrogen, or C1 to C3 alkyl;
R9 is C1 to C6 alkyl, C3 to C6 alkenyl, OH, NHR10, N(R10)2, CH2COR11, xe2x80x94CH2CHxe2x95x90CHCOR11, or 
R10 is C1 to C3 alkyl, C3 to C4 alkenyl, phenyl, xe2x80x94CH2CH2OCH3, or 
R11 is xe2x80x94OR12, xe2x80x94NHR12, xe2x80x94N(R12)2, or xe2x80x94NHCH2CH2CH2OR12;
R12 is hydrogen, or C1 to C3 alkyl;
R13 is hydrogen, or C1 to C3 alkyl;
W is a bond, CH2, CH2CH2, O, S(O)q, NCHO, NCOCH3, or NR12;
m is 0 -2;
n is 0-2;
q is 0-2,
with the proviso that R2 and R3 are not both hydrogen;
or pharmaceutically acceptable salt thereof.
The compounds of this invention antagonize the binding of hFSH to its receptor, in vitro, and to block cellular functions of FSH, in vitro, including the production of second messenger cAMP and estradiol in ovarian and granulosa cells. The compounds of this invention also inhibit FSH stimulated ovarian and uterine weight gain in immature female rats and ovulation in mature female rats. The compounds of this invention are useful as female and male contraceptive agents.
Preferred compounds of formula I are those in which:
X is a bond, S(O)n, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CH2CH2xe2x80x94, or xe2x80x94CH2S(O)nCH2xe2x80x94;
R1 is OH, or C1 to C6 alkoxy;
R2 and R4 are each, independently, hydrogen, OR6, xe2x80x94S(O)mR6, xe2x80x94NHR6, or xe2x80x94N(R6)2;
Y1 and Y3 are CH;
Z is xe2x80x94OR8 or xe2x80x94OCH2CH2OR8;
R9 is NHR10, N(R10)2, CH2COR11, xe2x80x94CH2CHxe2x95x90CHCOR11, or 
R10 is C1 to C3 alkyl, C3 to C4 alkenyl, xe2x80x94CH2CH2OCH3, or 
W is a bond, CH2, CH2CH2, O, S(O)q, NCHO, or NCOCH3;
or a pharamaceutically acceptable salt thereof, with the remaining substituents as defined above.
Specifically compounds in this invention include:
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(methylthio)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2-[(E)-1,2-ethenediyl]bis[5-[[4-(methylthio)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid], bis(1-methylethyl) ester;
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-(4-morpholinylsulfonyl)-4-[(tetrahydro-2H-pyran-4-yl)oxy]benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(2-methoxyethoxy)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid;
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(methylsulfonyl)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-[2-(2-methoxyethoxy)ethylthio]-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(methylsulfonyl)-3-nitrobenzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(methylsulfonyl)-3-nitrobenzoyl]amino]benzenesulfonic acid], bis(1-methylethyl) ester;
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-[(4-formyl-1-piperazinyl)sulfonyl)-4-methoxybenzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-[2-(2-methoxyethoxy)ethoxy]-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-methoxy-3-[[[2-(4-morpholinyl)ethyl]amino]sulfonyl]benzoyl]aminolbenzenesulfonic acid];
5-[[4-methoxy-3-(4-morpholinylsulfonyl)benzoyl]amino]-2-[(E)-2-[4-[[4-(methylsulfonyl)benzoyl]amino]-2-sulfophenyl]ethenyl]benzenesulfonic acid];
5-[[4-methoxy-3-[[(2-methoxyethyl)amino]sulfonyl]benzoyl]amino]-2-[(E)-2-[4-[[4-(methylsulfonyl)-3-nitrobenzoyl] amino]-2-sulfophenyl]ethenyl]benzenesulfonic acid];
5-[[4-methoxy-3-(4-morpholinylsulfonyl)benzoyl]amino]-2-[2-[4-[[4-(methylsulfonyl)benzoyl]amino]-2-sulfophenyl]ethyl]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-[(1,1-dioxido-4-thiomorpholinyl)sulfonyl]-4-methoxybenzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)1,2-ethenediyllbis[5-[[4-methoxy-3-[[(2-methoxyethyl)amino]sulfonyl]benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-[[bis(2-methoxyethyl)amino]sulfonyl]-4-methoxybenzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethanediyl]bis[5-[[4-methoxy-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
5-[[4-(methylsulfonyl)-3-nitrobenzoyl]amino]-2-[(E)-2-[4-[[4-(methylthio)-3-(4-morpholinylsulfonyl)benzoyl]amino]-2-sulfophenyl]ethenyl]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(ethylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(ethylsulfonyl)benzoyl]amino]benzenesulfonic acid], bis(1-methylethyl) ester;
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(methoxy)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(2-propenylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(ethylthio)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-[(4-acetyl-1-piperazinyl)sulfonyl]-4-methoxybenzoyl]amino]benzenesulfonic acid;
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-[(2-ethoxyethyl)amino]-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(methylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-amino-4-(methylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-[(2-methoxyethyl)thio]-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-methoxy-3-(1-pyrrolidinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(methylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethanediyl]bis[5-[[3-[(1,1-dioxido-4-thiomorpholinyl)sulfonyl]-4-methoxybenzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-(4-morpholinylmethyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(dimethylamino)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-[bis(2-methoxyethyl)amino]-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-(1,2-ethanediyl)bis[5-[[4-(2-methoxyethoxy)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-methoxy -3-(4-thiomorpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
5-[[4-(methylsulfonyl)benzoyl]amino]-2-[(E)-2-[4-[[4-[(methylsulfonyl)methyl]benzoyl]amino]-2-sulfophenyl]ethenyl]benzenefulfonic acid;
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-[(methylsulfonyl)methyl]benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-[(diethylamino)sulfonyl]-4-methoxybenzoyl]amino]benzenesulfonic acid];
5-[[4-(2-methoxyethoxy)benzoyl]amino]-2-[(E)-2-[4-[[4-(methylsulfonyl)benzoyl]amino]-2-sulfophenyl]ethenyl]benzenesulfonic acid;
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
5-[[4-(2-methoxyethoxy)benzoyl]amino]-2-[(E)-2-[4-[[4-methoxy-3-(4-morpholinylsulfonyl)benzoyl]amino]-2-sulfo-phenyl]ethenyl]benzenesulfonic acid;
2,2xe2x80x2-[1,2-ethanediyl]bis[5-[[4-methoxy-3-(1-pyrrolidinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(4-morpholinyl)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-methoxy-3-(1-piperidinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
5-[[4-(2-methoxyethoxy)benzoyl]amino]-2-[2-[4-[[4-methoxy-3-(4-morpholinylsulfonyl)benzoyl]amino]-2-sulfophenyl]ethyl]benzenesulfonic acid;
2,2xe2x80x2-(-1,2-ethanediyl)bis[5-[[3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[3-[(hexahydro-1H-azepin-1-yl)sulfonyl]-4-methoxybenzoyl]amino]benzenesulfonic acid;
2,2xe2x80x2-(1,2-ethanediyl)bis[5-[[4-methoxy-3-(1-piperidinylsulfonyl)benzoyl]amino]benzenesulfonic acid;
4,4xe2x80x2-bis[4-methoxy-3-(morpholine-4-sulfonyl)benzoylamino]biphenyl-2,2xe2x80x2-(bis)sulfonic acid;
2,2xe2x80x2-thiobis[5-[[4-methoxy-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid;
2,2xe2x80x2-[thiobis(methylene)]bis[5-[[4-methoxy-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[sulfonylbis(methylene)]bis[5-[[4-methoxy-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[[5-(methylsulfonyl)2-thienyl]carbonyl]amino]benzenesulfonic acid];
[4-(4-{2-[4-(4-methoxycarbonylmethanesulfonyl-benzoylamino)-2-sulfo-phenyl]-vinyl}-3-sulfo-phenylcarbomoyl)-benzenesulfonyl]-acetic acid, methyl ester;
[4-(4-{2-[4-(4-carboxymethanesulfonyl-benzoylamino)-2-sulfo-phenyl]-vinyl-)-3-sulfo-phenylcarbamoyl)-benzenesulfonyl]-acetic acid;
5-[[4-[[2-[(2-hydroxyethyl)amino]-2-oxoethyl]sulfonyl]benzoyl]amino]-2-[(E)-2-[4-[[4-[[2-[(2-hydroxyethyl)amino]-2-oxoethyl]sulfonyl]benzoyl]amino]-2-sulfo-phenyl]ethenyl]benzenesulfonic acid;
5-[[4-[(2-amino-2-oxoethyl)sulfonyl]benzoyl]amino]-2-[(E)-2-[4-[[4-[(2-amino-2-oxoethyl)sulfonyl]benzoyl]amino]-2-sulfophenyl]ethenyl]-benzenesulfonic acid;
[3-(4-{2-[4-(3-methoxycarbonylmethanesulfonyl-benzoylamino)-2-sulfo-phenyl]-vinyl}-3-sulfo-phenylcarbomoyl)-benzenesulfonyl]-acetic acid, methyl ester;
4-{3-[4-(2-{4-{3-(3-methoxycarbonyl-prop-2-ene-1-sulfonyl)-benzoylamino]-2-sulfo-phenyl}-vinyl)-3-sulfo-phenylcarbomoyl]-benzenesulfonyl}-but-2-enoic acid, methyl ester;
[3-(4-{2-[4-(3-carboxymethanesulfonyl-benzoylamino)-2-sulfo-phenyl]-vinyl}-3-sulfo-phenylcarbamoyl)-benzenesulfonyl]-acetic acid;
[4-[4-(2-{4-[4-methoxycarbonylmethylsulfanyl-3-(morpholine-4-sulfonyl)-benzoylamino]-2-sulfo-phenyl}-(E)-vinyl)-3-sulfo-phenylcarbamoyl]-2-(morpholine-4-sulfonyl)-phenylsulfanyl]-acetic acid, methyl ester;
4-{3-[4-(2-{4-[3-(3-carboxy-prop-2-ene-1-sulfonyl)-benzoylamino]-2-sulfo-phenyl}-vinyl)-3-sulfo-phenylcarbomoyl]-benzenesulfonyl}-but-2-enoic acid;
5-[[3-[(2-amino-2-oxoethyl)sulfonyl]benzoyl]amino]-2-[(E)-2-[4-[[3-[(2-amino-2-oxoethyl)sulfonyl]benzoyl]amino]-2-sulfophenyl]ethenyl]-benzenesulfonic acid;
5-[[3-[[2-[(2-hydroxyethyl)amino]-2-oxoethyl]sulfonyl]benzoyl]amino]-2-[(E)-2-[4-[[3-[[2-[(2-hydroxyethyl)amino]-2-oxoethyl]sulfonyl]benzoyl]amino]-2-sulfo-phenyl]ethenyl]benzenesulfonic acid;
2,2xe2x80x2-(1,2-ethanediyl)bis[5-[[4-(tetrahydro-2-furanmethyl)-3-(4-morpholinylsulfonyl)benzoyl]amino]benzenesulfonic acid];
2,2xe2x80x2-[(E)-1,2-ethenediyl]bis[5-[[4-(2-furanylmethoxy)-3-(4-morpholinylsulfony)benzoyl]amino]benzenesulfonic acid];
N-[3-(aminosulfonyl)-4-[(E)-2-[2-(aminosulfonyl-4-[[4-(methylsulfanyl)-3-(4-morpholinyl-sulfonyl)benzoyl]amino]phenyl]ethenyl]phenyl]-4-(methylsulfanyl)-3-(4-morpholinylsulfonyl)benzamide; and
5-[4-methylsulfanyl-3-(morpholine-4-sulfonyl)-benzoylamino]-2-(2-{[4-methylsulfanyl-3-(morpholine-4-sulfonyl)-benzoylamino]-phenyl}-vinyl)-benzenesulfonic acid;
and pharmaceutically acceptable salts thereof.
Pharmaceutically acceptable salts of the sulfonic acid residues of the compounds of formula (I) can be formed from organic and inorganic bases. For example alkali metal salts: sodium, lithium, or potassium and tetra-alkylammonium salts such as tetra-N-butylammonium salts. Similarly, when a compound of this invention contains a carboxylate or phenolic moiety, salts can be formed form organic and inorganic bases. Salts can also be formed from organic and inorganic acids, for example, acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable acids when a compound of this invention contains a basic moiety.
The compounds of this invention may contain an asymmetric carbon atom and some of the compounds of this invention may contain one or more asymmetric centers and may thus give rise to optical isomers and diastereomers. While shown without respect to stereochemistry in Formula (I), the present invention includes such optical isomers and diastereomers; as well as the racemic and resolved, enantiomerically pure R and S stereoisomers; as well as other mixtures of the R and S stereoisomers and pharmaceutically acceptable salts thereof.
The term xe2x80x9calkylxe2x80x9d is used herein to refer to both straight- and branched-chain saturated aliphatic hydrocarbon groups having at least one carbon atoms; xe2x80x9calkenylxe2x80x9d is intended to include both straight- and branched-chain alkyl group with at least one carbon-carbon double bond. The term xe2x80x9clower,xe2x80x9d when used in conjunction with alkyl, alkoxy and the like, indicates less than 6 carbon atoms. This invention covers both the E and Z conformations of such alkenyl moieties, with the E conformation being preferred. The term xe2x80x9cperfluoroalkylxe2x80x9d is used herein to refer to both straight- and branched-chain saturated aliphatic hydrocarbon groups having at least one carbon atom and two or more fluorine atoms. Examples include CF3, CH2CF3, CF2CF3 and CH(CF3)2.
The compounds of this invention can be prepared according to standard chemical methodology described in the literature from either commercially available starting material, or starting material which can be prepared as described in the literature. The compounds of this invention can be prepared according to the following synthetic schemes. Unless otherwise noted, Q, Ar, Arxe2x80x2, R1 to R13, W, X, Y1 to Y4, Z, q, n, and m are defined above. 
According to Scheme A, compound 1 can be reacted with one or slightly more than one equivalent of an acid chloride 2 (X2=Cl) to produce the compound 3. This reaction is usually performed in the presence of one or more equivalents of a organic amine base such as diisopropylethyl amine or one or more equivalents of an inorganic base such as sodium bicarbonate. Suitable solvents for this transformation include halocarbon solvents such as dichloromethane, THF, dioxane, dimethylacetamide or DMF. Water may be a co-solvent in this process. This reaction is usually performed in the temperature range including 0 to 160xc2x0 C. over a period of 30 minutes to 48 hours. The acid chloride 2 (X2=Cl) is readily prepared from acid 2 (X2=OH). For example, treatment of the acid 2 (X2=OH) with one or more equivalents of oxalyl chloride in the presence of a catalytic amount of DMF in a halocarbon solvent, such as dichloromethane, at temperatures ranging from 0 to 35xc2x0 C. will afford acid chloride 2 (X2=Cl).
Alternatively, the compound 3 can be prepared from the compound 1 and the acid 2 (X2=OH) using standard amidation and peptide coupling conditions. For instance, treatment of the acid 2 (X2=OH) with one or more equivalents of a commercially available carbodiimide such as dicyclohexylcarbodimide (DCC) or 1-(3-dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI) and subsequent reaction with the compound 1 results in the formation of the compound 3. The reaction is conveniently performed with or without one or more equivalents of commercially available additive N-hydroxybenzotriazole (HOBT). and with or without one or more equivalents of an organic base such as triethylamine or diisopropylethylamine or an inorganic base such as sodium bicarbonate. Solvents generally useful include halocarbon solvents such as dichlormethane, THF or DMF.
Treatment of compound 3 with one or more equivalents of acid chloride 4 (X2=Cl) or the acid 4 (X2=OH) using conditions described above for the 1 to 3 transformation results in the formation of compound 5. When Ar=Arxe2x80x2 for compound 5, compound 5 can be made directly from compound 1, by employing two or more equivalents of acid chloride 2 (X2=Cl) or acid 2 (X2=OH) and otherwise following the process for the 1 to 3 transformation.
When R1 is OH, the above acylation transformations are most conveniently done by converting the sulfonic acid moieties of 1, 3, or 5 to their tetrabutylammonium salt forms (R1=ONBu4). This conversion is done by treating an aqueous THE solution of 1, 3, or 5 with two or more equivalents of tetrabutylammonium hydroxide at ambient temperatures.
When R1 is not H in the compound 5, this compound can be deesterified to provide the compound 6. This is most conveniently accomplished using one or more molar equivalents of a alkali metal or tetraalkylammonium halide such as sodium iodide, lithium bromide, or tetrabutylammonium chloride in a suitable solvent such as acetone, 2-butanone or DMF with or without a co-solvent such as water at temperatures ranging from 0 to 130xc2x0 C. and over a time period of one to 48 h. Other methods to effect deesterification to the compound 6 include reacting the compound 5 with one or more equivalents of an organic base such as piperidine and dimethylaminopyridine in an organic solvent such as THF or DMF at temperatures ranging from 20 to 120xc2x0 C. over periods of 1 h to 64 h. 
According to Scheme B, the compound 1 can be protected as its mono-BOC derivative 7 (BOC=COOtBu), using standard methods. For example, the compound 1 can be reacted with one equivalent of di-t-butyl-dicarbonate in a THF, dioxane or DMF at temperatures ranging from 0 to 40xc2x0 C. to afford the BOC protected compound 7. Treatment of compound 7 with one or more equivalents of acid chloride 2 (X2=Cl) or the acid 2 (X2=OH) using conditions described above for the 1 to 3 transformation in Scheme A results in the formation of compound 8. The BOC group of compound 8 can be removed to produce the monoacylated compound 3 using standard conditions. For example, compound 8 can be reacted with one or more equivalents of trifluoroacetic acid in a haloform solvent or using the trifluoroacetic acid as the solvent to provide the compound 3. Compound 3 can be further elaborated as shown in Scheme A. 
According to Scheme C, the compound 9 can be protected as its FMOC derivative 10 (FMOC=9-fluorenylmethoxycarbonyl) using standard methods. For example, the compound 9 can be treated with one or more equivalents of an alkali metal carbonate, such as sodium carbonate, and two or more equivalents of 9-fluorenylmethyl chloroformate in a lower alcohol solvent such as methanol or a mixture of dioxane/water at temperature ranging from 0 to 40xc2x0 C. to afford the FMOC protected compound 10.
The compound 10 can then be esterified on the sulfonic acid moiety to the ester 11 using a procedure similar to sulfonic acid esterification methods of A. A Padmapriya, G. Just and N. G. Lewis Synthetic. Comm. 1985, 15, 1057-1062 and J. I. Trujillo and A. S. Gopalan Tetrahedron Lett. 1993, 34, 7355-7358 employing the commercially available tri-alkylorthoformate [HC(R1)3] as the esterification reagent. The acid form of the compound 10 is heated with one or more equivalents of the tri-alkylorthoformate in a suitable solvent such as dioxane at temperatures ranging from 40 to 100xc2x0 C. over a period ranging from one to 48 h to produce the ester 11.
The FMOC group of the compound 11 can be removed using standard conditions, most notably using one or more equivalents of an organic amine base such as piperidine in a suitable solvent such as DMF or THF to provide the amine 1 (R1=lower alkoxy). This reaction is most conveniently done at the temperature range of 0 to 40xc2x0 C. over a time period of 5 minutes to 10 h. The compound 1 (R1=lower alkoxy) can then be elaborated according to Scheme A. 
According to Scheme D, the compound 12 can be esterified on the sulfonic acid moiety to the ester 13 using a procedures outlined in Scheme C, employing a commercially available tri-alkylorthoformate [HC(OR1)3] as the esterification reagent. The nitro groups of ester 13 can be reduced to amino moieties of compound using a variety of standard reducing agents, including, but not limited to, catalytic hydrogenation using a palladium or platinum catalyst, tin chloride in aqueous HCl, ethyl acetate, ethanol, dioxane, THF, or DMF solvents, sodium sulfide in aqueous lower alcohol solvent, and hydrazine and Montmorillinite clay in ethanol. The compound 1 (R1=lower alkoxy) can then be elaborated according to Scheme A. 
Scheme E illustrates an alternative preparation of stilbene (bis)sulfonic acid subset of the compounds of formula (I). According to Scheme E, the commercially available sulfonyl chloride 14 can be reacted with one or more equivalents of a commercially available alcohol or ammonia under standard conditions to afford the sulfonic acid ester or amide 15 (R1=lower alkoxy, NH2). Standard conditions include, but are not limited to, the use of one or more equivalents of a tertiary amine base such as triethylamine, or pyridine, or an alkali metal carbonate or hydroxide such as sodium carbonate or potassium hydroxide in solvents which can include water, halocarbon, lower alcohol, THF or dioxane, at temperatures ranging from 0 to 80xc2x0 C. over a time period of 5 minutes to 12 h.
The sulfonic acid derivative 15, can be treated with one or more equivalents of potassium t-butoxide in DMF in the presence of air at 0xc2x0 C. to ambient temperatures to afford the stilbene analog 16a. The nitro groups of 16a can be reduced to amino compound 16b using a variety of standard reducing agents, including, but not limited to, tin chloride in aqueous HCl, ethyl acetate, ethanol, dioxane, THF, or DMF solvents, sodium sulfide in aqueous lower alcohol solvent, and hydrazine and Montmorillinite clay in ethanol. The compound 16b can then be elaborated according to Scheme A. 
Scheme F illustrates a preparation of thio(bismethylene)-(bis)sulfonic acid subset of the compounds of formula (I). According to Scheme F, the compound 15 can be brominated on the benzylic carbon using to the bromide 17. Typically this is most conveniently done using one equivalent of N-bromosuccinimide (NBS) and a catalytic amount of benzoyl peroxide in an inert solvent such as carbon tetrachloride or dichloromethane at temperatures ranging from 0 to 60xc2x0 C. over a time period of 30 minutes to 48 h. The bromide 17 can be treated with one or more equivalents of thioacetamide in chloroform at temperatures ranging from from 0 to 60xc2x0 C. over a time period of 30 minutes to 48 h to afford, after aqueous workup, thiol 18.
The compound 18, can be reacted with the compound 17 using one or more equivalents of a base promoter, such as triethylamine, or potassium carbonate in an inert solvent such as THF, dichloromethane or acetonitrile at temperatures ranging from 0 to 60xc2x0 C. over a time period of 30 minutes to 48 h to afford the compound 19. The nitro groups of 19 can be reduced to amino compound 20 using a variety of standard reducing agents, including, but not limited to, catalytic hydrogenation using a palladium or platinum catalyst, tin chloride in aqueous HCl, ethyl acetate, ethanol, dioxane, THF, or DMF solvents, sodium sulfide in aqueous lower alcohol solvent, and hydrazine and Montmorillinite clay in ethanol. The compound 20 can then be elaborated according to Scheme A. 
A, B are independent and equal to NH2, NH(FMOC), NH(BOC), NHCOAr, NHCOArxe2x80x2
Scheme G illustrates a preparation of dihydrostilbene (bis)sulfonic acid subset of the compounds of formula (I). According to Scheme G, the double bond of stilbene 21, can be reduced most conveniently by catalytic hydrogenation using a palladium or platinum catalyst in aqueous alcohol to afford the dihydrostilbene 22. Compound 22 [A and/or B is NH2, NH(FMOC), NH(BOC)] can be further elaborated according to Schemes A-D. 
According to Scheme H, the thioethers 23a or 23b can be converted to their sulfone derivatives 24a or 24b respectively using two or more molar equivalents of an oxidizing agent such as oxone in an aqueous alcohol solvent at temperatures ranging from room temperature to 100xc2x0 C., m-chloroperbenzoic acid in dichloromethane at temperatures ranging from xe2x88x9220xc2x0 C. to 60xc2x0 C. or hydrogen peroxide in acetic acid at temperatures ranging from room temperature to 100xc2x0 C. Compounds 24a or 24b [A and/or B is NH2, NH(FMOC), NH(BOC)] can be further elaborated according to Schemes A-D.
The carboxylic acids, ArCO2H and Arxe2x80x2CO2H are prepared according to Schemes I through L. 
Scheme I illustrates the preparation of benzoic acid analogs that can be used in the preparation of target compounds outlined in Scheme""s A and B. Acid derivative 25 can be treated with neat chlorosulfonic acid at temperatures ranging from xe2x88x9220xc2x0 C. to 150xc2x0 C. to afford the sulfonyl chloride derivatives 26. The compounds 26 can be treated with a variety of primary or secondary amines in an inert solvent such as dichloromethane or THF at temperatures ranging from 0xc2x0 C. to 50xc2x0 C. to provide the sulfonamide derivatives 27.
Treatment of 27 [E=F (fluorine)] with nucleophiles such as alkoxides (OR6), thiolates (SR6), and amines (NH2R6, NH(R6)2) affords compounds 28 in which the nucleophilic moiety replaces the fluoride atom of 27. Solvents for this reaction include water, THF, dioxane, DMF, acetonitrile, dichloromethane, lower alcohol, or combinations of these solvents. The reactions can be conveniently performed at temperatures ranging from xe2x88x9220xc2x0 C. to 150xc2x0 C. over a 5 minute to 48 hr period. The alkoxide or thiolate nucleophiles are generally prepared in situ by treating one or more equivalents of the corresponding alcohol (HOR6) or thiol (HSR6) with more than one equivalent amount of a base such as sodium hydride, butyl lithium, potassium carbonate or triethylamine. When amines (NH2R6, NH(R6)2) are used as nucleophiles, one or more equivalents of these reagents are used.
When Nu of 28 is SR6, the thioether sulfour atom of 28 can be oxidized to the sulfone 29. This oxidation is most conveniently done using two or more molar equivalents of an oxidizing agent such as oxone in an aqueous alcohol solvent at temperatures ranging from room temperature to 100xc2x0 C., m-chloroperbenzoic acid in dichloromethane at temperatures ranging from xe2x88x9220xc2x0 C. to 60xc2x0 C. or hydrogen peroxide in acetic acid at temperatures ranging from room temperature to 100xc2x0 C.
The reactions for compounds 25-29 can generally be carried out on the acid form (Rc=H) or the ester form (Rc is lower alkyl). For the target products of Scheme I (compounds 27-29) to be properly utilized in Schemes A and B, esters of 27-29 (Rc is lower alkyl) must be converted to their acid forms (Rc=H). The conditions to most conveniently effect these transformations include aqueous base in which one or more molar equivalents of alkali metal hydroxide such as sodium, lithium or potassium hydroxide is used in water with a co-solvent such as THF, dioxane or a lower alcohol such as methanol or mixtures of THF and a lower alcohol at temperatures ranging from 0xc2x0 C. to 40xc2x0 C. 
Scheme J further illustrates the preparation of benzoic acid analogs that can be used in the preparation of target compounds outlined in Scheme""s A and B. Compound 31 can be prepared from compound 30 via a two-step, one pot reaction. In this regard, compound 30 can be reacted with the sodium salt of 2-(methylsulfonyl)ethanol. The alcohol moiety of 2-(methylsulfonyl)ethanol displaces the fluorine atom of 30. Subsequently, in situ, or during aqueous workup, vinyl-methylsulfone is released from the 30/2-(methylsulfonyl)ethanol adduct via an E2 type of elimination reaction to afford the phenol 31. This reaction is most conveniently done employing one or more equivalents of 2-(methylsulfonyl)ethanol and three or more equivalents of sodium hydride as the base. The reaction can be performed within the temperature range of 0xc2x0 C. to 40xc2x0 C., within a 5 min to 12 h period. Suitable solvents include DMF, THF, dioxane, and acetonitrile.
The phenol 31 can be alkylated with one or more molar equivalents of an alkyl halide, tosylate, mesylate or triflate (R6X3, X3 is Cl, Br, I, OSO2Ph, OSO2CH3, OSO2CF3) using one or more molar equivalents of an alkali metal carbonate such as potassium carbonate or one or more equivalent of an alkali metal hydride such as sodium hydride in a polar aprotic solvent such as DMF to afford the alkylated phenol 32. Alternatively, the phenol 31 can be reacted with an alcohol R6OH to afford compound 32 under the conditions of the Mitsunobu reaction. The other co-reagents necessary to effect the Mitsunobu reaction include one or more molar equivalents of an alkyl of 1-6 carbon atoms azodicarboxylate diester such as diethyl azodicarboxylate or diisopropyl azodicarboxylate and one or more molar equivalents of triarylphosphine such as triphenylphosphine in a suitable solvent such as diethyl ether, THF, benzene or toluene at temperatures ranging from xe2x88x9220xc2x0 C. to 120xc2x0 C.
The reactions for compounds 32 can generally be carried out on the acid form (Rc=H) or the ester form (Rc is lower alkyl). For the target products of Scheme J (compound 32) to be properly utilized in Schemes A and B, esters of 32 (Rc is lower alkyl) must be converted to their acid forms (Rc=H). The conditions to most conveniently effect these transformations include aqueous base in which one or more molar equivalents of alkali metal hydroxide such as sodium, lithium or potassium hydroxide is used in water with a co-solvent such as THF, dioxane or a lower alcohol such as methanol or mixtures of THF and a lower alcohol at temperatures ranging from 0xc2x0 C. to 40xc2x0 C. 
Scheme K further illustrates the preparation of benzoic acid analogs that can be used in the preparation of target compounds outlined in Scheme""s A and B. The thiophenol 33 can be alkylated with one or more molar equivalents of an alkyl halide, tosylate, mesylate or triflate (R6X3, X3 is Cl, Br, I, OSO2Ph, OSO2CH3, OSO2CF3) using one or more molar equivalents of an alkali metal carbonate or hydroxide such as potassium carbonate or potassium hydroxide, one or more equivalent of an alkali metal hydride such as sodium hydride or one or more equivalents of a tertiary amine such as triethylamine in a polar aprotic solvent such as DMF or in an lower alcohol solvent such as ethanol or a halocarbon solvent such as dichloromethane to afford the thioether 34. Alternatively, the thiophenol 33 can be reacted with an alcohol R6OH to afford compound 34 under the conditions of the Mitsunobu reaction. The other co-reagents necessary to effect the Mitsunobu reaction include one or more molar equivalents of a alkyl of 1-6 carbon atoms azodicarboxylate diester such as diethyl azodicarboxylate or diisopropyl azodicarboxylate and one or more molar equivalents of triarylphosphine such as triphenylphosphine in a suitable solvent such as diethyl ether, THF, benzene or toluene at temperatures ranging from xe2x88x9220xc2x0 C. to 120xc2x0 C.
The thioether sulfur atom of 34 can be oxidized to the sulfone 35. This oxidation is most conveniently done using two or more molar equivalents of an oxidizing agent such as oxone in an aqueous alcohol solvent at temperatures ranging from room temperature to 100xc2x0 C., m-chloroperbenzoic acid in dichloromethane at temperatures ranging from xe2x88x9220xc2x0 C. to 60xc2x0 C. or hydrogen peroxide in acetic acid at temperatures ranging from room temperature to 100xc2x0 C.
The reactions for compounds 35 can generally be carried out on the acid form (Rc=H) or the ester form (Rc is lower alkyl). For the target products of Scheme K (compound 35) to be properly utilized in Schemes A and B, esters of 35 (Rc is lower alkyl) must be converted to their acid forms (RcH). The conditions to most conveniently effect these transformations include aqueous base in which one or more molar equivalents of alkali metal hydroxide such as sodium, lithium or potassium hydroxide is used in water with a co-solvent such as THF, dioxane or a lower alcohol such as methanol or mixtures of THF and a lower alcohol at temperatures ranging from 0xc2x0 C. to 40xc2x0 C. 
Scheme L illustrates the preparation of sulfonyl-substituted thiophene carboxylic acid analogs that can be used in the preparation of target compounds outlined in Scheme""s A and B. The thioether sulfur atom of 36 can be oxidized to the sulfone 37. This oxidation is most conveniently done using two or more molar equivalents of an oxidizing agent such as oxone in an aqueous alcohol solvent at temperatures ranging from room temperature to 100xc2x0 C., m-chloroperbenzoic acid in dichloromethane at temperatures ranging from xe2x88x9220xc2x0 C. to 60xc2x0 C. or hydrogen peroxide in acetic acid at temperatures ranging from room temperature to 100xc2x0 C.
The reactions for compounds 37 can generally be carried out on the acid form (Rc=H) or the ester form (Rc is lower alkyl). For the target products of Scheme L (compound 37) to be properly utilized in Schemes A and B, esters of 37 (Rc is lower alkyl) must be converted to their acid forms (Rc=H). The conditions to most conveniently effect these transformations include aqueous base in which one or more molar equivalents of alkali metal hydroxide such as sodium, lithium or potassium hydroxide is used in water with a co-solvent such as THF, dioxane or a lower alcohol such as methanol or mixtures of THF and a lower alcohol at temperatures ranging from 0xc2x0 C. to 40xc2x0 C. 
Scheme M illustrates a preparation of stilbene (mono)sulfonic acid subset of the compounds of formula (I). According to Scheme M, bromide 17 can be reacted with one or more equivalents of triethylphosphite to afford the phosphonate 38. The transformation 17 to 38 is known as the Arbuzov reaction. Typically this reaction is done in an inert solvent such as dichloromethane, acetonitrile, DMF or THF at temperatures ranging from room temperature to 150xc2x0 C.
Compound 38 can undergo a Homer-Emmons condensation reaction with one or more equivalents of p-nitrobenzaldehyde to afford the stilbene 39. One or more equivalents of a base is used to promote this reaction. Typical bases include butyl lithium, potassium hydroxide, potassium t-butoxide and sodium hydride. Typically this reaction is done in an inert solvent such as a lower alcohol, DMF or THF at temperatures ranging from 0xc2x0 C. to 150xc2x0 C.
The nitro groups of compound 39 can be reduced to amino moieties of compound 40 using a variety of standard reducing agents, including, but not limited to, catalytic hydrogenation using a palladium or platinum catalyst, tin chloride in aqueous HCl, ethyl acetate, ethanol, dioxane, THF, or DMF solvents, sodium sulfide in aqueous lower alcohol solvent, and hydrazine and Montmorillinite clay in ethanol. Compound 40 can be reacted with two or more equivalents of an acid chloride 2 (X2=Cl) to produce the compound 41. This reaction is usually performed in the presence of two or more equivalents of a organic amine base such as diisopropylethyl amine or one or more equivalents of an inorganic base such as sodium bicarbonate. Suitable solvents for this transformation include halocarbon solvents such as dichloromethane, THF, dioxane, dimethylacetamide or DMF. Water may be a co-solvent in this process. This reaction is usually performed in the temperature range including 0 to 160xc2x0 C. over a period of 30 minutes to 48 hours. The acid chloride 2 (X2=Cl) is either commercially available or readily prepared from commercially available acid 2 (X2=OH). Standard reagents and conditions are used to effect the acid to acid chloride transformation, for example, treatment of the acid 2 (X2=OH) with one or more equivalents of oxalyl chloride in the presence of a catalytic amount of DMF in a halocarbon solvent, such as dichloromethane, at temperatures ranging from 0 to 35xc2x0 C. will afford acid chloride 2 (X2=Cl).
Alternatively, the compound 41 can be prepared from the compound 40 and the acid 2 (X2=OH) using standard amidation and peptide coupling conditions. For instance, treatment of the acid 2 (X2=OH) with two or more equivalents of a commercially available carbodiimide such as dicyclohexylcarbodimide (DCC) or 1-(3-dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI) and subsequent reaction with the compound 40 results in the formation of the compound 42. The reaction is conveniently performed with or without one or more equivalents of commercially available additive N-hydroxybenzotriazole (HOBT). and with or without one or more equivalents of an organic base such as triethylamine or diisopropylethylamine or an inorganic base such as sodium bicarbonate. Solvents generally useful include halocarbon solvents such as dichlormethane, THF or DMF. 
Scheme N illustrates further ellaboration of certain bis(sulfonic) acid derivatives 42. The compounds 42 can be converted to the carboxylic acid forms 43 (Nu2=OH). The conditions to most conveniently effect these transformations include aqueous base in which one or more molar equivalents of alkali metal hydroxide such as sodium, lithium or potassium hydroxide is used in water with a co-solvent such as THF, dioxane or a lower alcohol such as methanol or mixtures of THF and a lower alcohol. at temperatures ranging from 0xc2x0 C. to 40xc2x0 C.
The compounds 42 can also be converted to the carboxylic acid amide forms 43 (Nu2=R11NH, (R11)2N). The conditions used to effect this transformation include reaction of 42 with one or more equivalents of a primary or secondary amine (HNu2=R11NH2, (R11)2NH) in a suitable solvent such as a lower alcohol solvent, THF or DMF at temperatures ranging from 0xc2x0 C. to 120xc2x0 C.
The FSH antagonist activities of the compounds of this invention were demonstrated by evaluating representative compounds of this invention in three in vitro FSH antagonist and one in vivo standard pharmacological test procedures.
FSH Receptor Radioligand Membrane Binding Standard Pharmacological Test Procedure
Membrane Source: Chinese hamster ovarian cells stably transfected with the human FSH receptor were cultured (Ultra CHO medium containing 1% fetal bovine serum and 200 xcexcg/mL G418) in and harvested. Cells were collected by centrifugation and resulting cell pellets were frozen and stored at xe2x88x9270xc2x0 C.
Membrane Receptor Preparation: Frozen cell pellets were weighed and resuspended in binding buffer to a final concentration of 30 mg wet weight/mL. Cell suspension for each pellet was homogenized using a Tempest homogenizer (setting=1; 5 strokes; VirTis, Gardiner, N.Y.). Cell homogenates were pooled and 35 mL aliquots were transferred to 50-mL polypropylene copolymer centrifuge tubes (Nalgene cat. #3110-0500). Tubes were spun at 12,000 rpm (SS34 rotor) for 12 min at 4xc2x0 C. Resulting supernatant fractions were discarded and pellets were stored at xe2x88x9270xc2x0 C. until use. On the day of the procedure, 35 mL binding buffer was added to each tube (35 mL membrane suspension was sufficient for three 96-well plates). The membrane pellet was dispersed by trituration using a pipet. The resulting suspension wass homogenized using a Tempest homogenizer (3 strokes at setting=1).
Receptor Binding Test procedure: Membrane homogenate (100 xcexcl) was added to each well of a 96-well microtiter plate (Falcon # 3077). All reactions were tested in triplicate. Test compound solutions (50 xcexcl) were added to the designated wells. Total bound counts were determined by adding 50 xcexcl binding buffer containing 4% DMSO to the designated wells. Non-specific binding was determined by adding 50 xcexcl of hFSH solution to the designated wells. Plates were pre-incubated for 15 min at room temperature on shaking platform (setting=3). After preincubation [125I]FSH (50 xcexcl) was added to each well and plates were incubated for 2 h at room temperature on shaking platform (setting=3). The reaction was terminated by transfer of the membrane preparation to glass fiber filters (Blue Mat #11740; 102xc3x97256 mm; Skatron Instruments, Sterling, Va.) that had been pretreated with 1% BSA in wash buffer for at least 30 min, but not longer than 1 h using a 96-well microtiter vacuum harvester (Skatron Instruments). The membrane preparation was washed with 5 cycles of ice-cold wash buffer (200 xcexcl/well/cycle) followed by a pulse wash of 3 cycles (100 xcexcl/well/cycle). The total wash volume per well was 1.3 mL. The filters were dried by a 10 sec aspiration. Disks corresponding to each well of the microtiter plate were punched out of the filter mat into 12xc3x9775 mm polypropylene tubes. The radioactivity present on each of the disks was measured using a gamma counter.
An FSH dose response curve (0.001, 0.01, 0.1, 1, 10, and 100 nM) was generated for each binding procedure to monitor procedure to procedrue variability.
FSH Receptor Radioligand Membrane Binding Procedure Buffers and Reagents
Binding buffer is prepared in 11 volumes containing Trizma-HCl, MgCl2, CaCl2 and sodium azide, the pH was adjusted to 7.2 with NaOH, and stored at 4xc2x0 C. until use. BSA was weighed out on the day of the procedure and added to the amount of buffer required for the procedure (usually 150 mL). The protease inhibitors were prepared as 1 mg/mL stocks (aprotinin, leupeptin, and phosphoramidon were prepared in binding buffer without BSA and protease inhibitors; pepstatin and PMSF were prepared in methanol), stored in 1 mL aliquots at xe2x88x9270xc2x0 C., and added to the binding buffer on the day of the procedure.
Wash buffer was prepared containing Trizma-HCl, MgCl2 and EDTA, the pH was adjusted to 7.2 with NaOH, and stored at 4xc2x0 C.
Filter Soak Buffer (pH 7.2)
Wash Buffer
1% BSA
BSA was weighed out on the day of the procedure and added to 300 mL of wash buffer. The filter soak buffer was used for two procedures before being discarded.
[125I]hFSH Solution: The concentration of the [125I]hFSH stock solution was determined by measuring the radioactivity in three 10 xcexcl samples of the stock solution using a gamma counter. The concentration was calculated using the radioactivity measurement (cpm), counting efficiency (0.8) to convert cpm to dpm and subsequent conversion of dpm to xcexcCi, specific activity xcexcCi/xcexcg FSH) of the [125I]hFSH given on the specification sheets from NEN, and the molecular weight of FSH (29,695). A portion of the stock solution was diluted in binding buffer to a concentration of 200 pM.
FSH Solution for Determining Non-specific Binding: Purified human FSH was prepared as a 100 xcexcM solution in binding buffer without protease inhibitors. This stock was stored as 30 xcexcl aliquots at xe2x88x9270xc2x0 C. The stock was diluted on the day of the procedure to 4 xcexcM in binding buffer containing 4% DMSO on the day of the procedure.
Compound Solutions: Each compound to be tested was prepared as a 400 xcexcM solution in DMSO. For additional concentrations, the 400 xcexcM stock solution was diluted with binding buffer containing 4% DMSO.
1) McPherson, G. A. 1985. Kinetic, EBDA, Ligand, Lowry: a collection of radioligand binding analysis programs. BIOSOFT, Cambridge, U. K.
2) Schneyer, A. L., Sluss, P. M., Bosukonda, D. and Reichert, L. E. xe2x80x9cElectrophoretic Purification of Radioiodinated Follicle-Stimulating Hormone for Radioligand Receptor Assay and Radioimmunoassay.xe2x80x9d Endocrinology, 1986, 119, 1446-1453.
3) Reichert, L. E. and Bhalla, V. K. xe2x80x9cDevelopment of a Radioligand Tissue Receptor Assay for Human Follicle-Stimulating Hormone.xe2x80x9d Endocrinology 1974, 94, 483-491.
The results obtained in this standard pharmaceutical test procedure are provided in the table below.
In Vitro Bio-test Procedure of Agonists and Antagonists to the FSH Receptor
Objective: This procedure was used to verify in vitro efficacy of compounds found to bind to the FSH receptor in the binding procedure.
Methods: Reagents
Compound Vehicle: Stock compounds were solubilized in an appropriate vehicle preferably PBS/0.1% Bovine Serum Albumin (BSA; Sigma Chemical Co., St. Louis, Mo.). The compounds were subsequently diluted in sterile procedure medium (Optimem (Gibco/BRL, Grand Island, N.Y.)/0.1% BSA) prior to use in the bio-procedure.
Preparation of CHO-3D2 Cells; CHO-3D2 cells were plated into 96-well Nunc tissue culture plates at a density of 30,000 cells/well in DMEM/F12 medium (Gibco/BRL, Grand Island, N.Y.) supplemented with 5% Fetal Bovine Serum (Hyclone, Fetal Clone II), 2 mM L-glutamine and penicillin/streptomycin (100 U/mL). Cells were plated one day prior to performing the bio-procedure.
Procedure: On the day of procedure, the wells were washed two times with 100 ul/well of pre-warmed (37 deg C.) procedure medium. After aspirating the second wash, an additional 100 ul of procedure medium was added to each well and the cells pre-incubated for 30-45 minutes at 37 deg C. In a humidified incubator with 5%CO2/95% air. The cells were then challenged with varying dilutions of the test substance(s) in a 50 ul total incubation volume in procedure medium for 30 minutes at 37 deg C. in the humidified incubator. The challenge was terminated by the addition of 50 ul of 0.2 N HCl to each well. CAMP accumulation in the medium was measured by radioimmunoassay.
Test Groups: In the 96-well format, the plate is organized into 12 columns each containing 8 rows of wells. The plate was split in half to test a single compound in both agonist and antagonist mode on the same plate.
For agonist mode, compounds were tested using 5 different concentrations in a dose-response paradigm using one column as a control (challenge medium alone) in agonist mode.
For antagonist mode, compounds were tested in a dose-response paradigm versus a constant level of purified human FSH (the ED20 (1.85 ng/mL); previously calculated during characterization of the bio-procedure). The 96-well format allowed for the capability to test 4 columns of compound, using one of the remaining columns for negative control (challenge medium alone) and the other remaining column for ampositive control (ED20 of FSH alone).
The doses chosen to test each compound were extrapolated from the initial screening process (receptor binding data). Along with the test compounds, FSH was run in agonist mode using doses ranging from 0.1 ng/mL-1000 ng/mL as a postive control. Cytotoxicity of the compounds were screened by treating cells with the highest concentration of each compound used in the cAMP procedure for 30 minutes followed by washing of the cells 2 times with 100 ul PBS. The cells were then incubated for 5 min at 37 deg C. in the presence of 50 ug/mL Fluorescein diacetate and 20 ug/mL Propidium iodide in 100 ul PBS. The cells were washed two times with 100 ul PBS followed by examination of the cells under a fluorescence microscope using a 490 nm filter. Viable cells stained green throughout, while dead cells had red fluorescent nuclei.
Analysis of Results: cAMP accumulation was expressed as fmol/mL. CAMP accumulation in agonist mode, or the ability of the compound to inhibit hFSH-induced cAMP accumulation in antagonist mode was compared to the appropriate negative and positive controls. Data were analyzed statistically by analysis of variance and significant differences between treatments and control determined by Dunnett""s test. In antagonist mode, a Duncan""s test was used.
Reference Compounds: Test compounds were compared to the effect of purified or recombinant human FSH. In this paradigm, hFSH induced a dose-dependent increase in cAMP accumulation, with apparent ED80=22.55 ng/mL, ED50=6.03 ng/mL and ED20=1.85 ng/mL, calculated using a four-parameter logistic equation.
The results obtained in this standard pharmacological test procedure are provided below.
In Vitro Bio-test Procedure of Agonists and Antagonists to the FSH Receptor Using Primary Cultures of Rat Granulosa
Objective: This procedure was used as a low-throughput functional screening procedure to study in vitro efficacy of compounds found to be agonists or antagonists of the FSH receptor.
Materials and Methods: Reagents
Compound Vehicle: Stock compounds were solubilized in an appropriate vehicle, preferably PBS (phosphate buffered saline) or DMSO (dimethyl sulfoxide), at a concentration of 0.1 M. The compounds were subsequently diluted in sterile challenge medium [McCoy""s 5A medium (Gibco/BRL, Grand Island, N.Y.) supplemented with 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL sodium selenite (ITS, Sigma Chemical Co., St. Louis, Mo.), 146 mg/mL L-glutamine, 100 nM testosterone, 100 nM DES and 100 U/mL penicillin/10 mg/mL streptomycin/250 ng/mL amphotericin B (antibiotic/antimycotic, Gibco) and 0.1% bovine serum albumin (Sigma, St. Louis, Mo.)] prior to use in the procedure. The concentration of vehicle was maintained constant throughout all dilutions.
Preparation of Granulosa Cells: Twenty-four day-old immature female Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass.) were used as donors for ovarian granulosa cells. The animals were treated by single daily injections of 100 mg/kg diethylstilbestrol (DES) in oil over three days. On the fourth day, animals were sacrificed by CO2 asphyxiation and the ovaries were removed. Ovaries were washed three times in 50 mL of sterile HEPES-buffered saline (HBS, pH 7.4). Granulosa cells were harvested by incubating ovaries in a hypertonic medium consisting of serum-free McCoy*s SA medium (Gibco Life Sciences, Grand Island, N.Y.) supplemented with 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL sodium selenite (ITS, Sigma Chemical Co., St. Louis Mo.), 146 mg/mL L-glutamine, 100 nM testosterone, 100 nM DES and 100 U/mL penicillin/10 mg/mL streptomycin/250 ng/mL amphotericin B (antibiotic/antimycotic, Gibco) containing 0.5 M sucrose and 0.1 mM EGTA. Ovaries were then incubated for 45 min. at 37 C. in a humidified incubator gassed with 95% air/5% CO2. They were washed 3 times with 10 mL isotonic medium (hypertonic medium without sucrose and EGTA) and incubated another 45 min. in isotonic medium at 37 C. Granulosa cells were harvested by squeezing the ovaries between two sterile glass microscope slides. Isolated granulosa cells were then placed in an 50 mL centrifuge tube and washed two times by the addition of 50 mL serum-free McCoy*s 5A medium followed by centrifugation at 700xc3x97g for 5 min. After the final spin, the cells were resuspended by gentle trituration in 25 mL serum-free medium, an aliquot counted in a hemocytometer and viability estimated by trypan blue exclusion. Cells were plated into 24-well Nunc tissue culture plates at 200,000 viable cells/well in 250 mL.
Procedure: Following plating of the cells, the plates are incubated at 37 C for 2-4 hours at which time the treatments are added to the cells. Treatments are added to the wells at 2xc3x97 the desired final concentration in 250 mL/well in isotonic medium containing 0.2% BSA. The cells are incubated at 37 C. for 72 h. At the end of the incubation period, the medium is removed from the wells and tested for estradiol concentration by radioimmunoassay.
Experimental Groups: In the 24-well format, the plate was divided into 6 columns of 4 wells/column. One plate per compound was used to test either agonist or antagonist modes.
In agonist mode, each compound was tested in a dose-response paradigm using 5 different doses of the compound and compared the activity to the 6th column of cells which received vehicle alone.
For antagonist mode, each compound was tested in a dose-response paradigm versus a constant level of purified human FSH (the ED50 0.5 ng/mL; previously calculated during the characterization of the bioprocedure). Four different doses of compound were tested in the antagonist mode. In addition, one column was used for a negative control (vehicle alone) and the other remaining column for a positive control (ED50 of FSH alone).
The doses of compound chosen to test were extrapolated from the initial functional screening process. Along with the plates testing compounds, another plate was run in parallel using a dose-response of FSH (0.01-100 ng/mL) as a positive control.
Analysis of Results: Estradiol was expressed as pg/mL. Estradiol secretion in agonist mode, or the ability of the compound to inhibit FSH-induced estradiol secretion in antagonist, was compared to the appropriate negative and positive controls. Data were analyzed statistically by analysis of variance with Huber weighting of log transformed data. Paired differences were determined using the LSD test
Reference Compounds: Test compounds were compared to the effect of purified or recombinant human FSH.
Activity: Compounds which significantly increase estradiol secretion as compared to the negative control in agonist mode or significantly inhibited FSH-induced estradiol secretion in antagonist mode were considered active. EC50: Concentration of the compound that gave half-maximal response in terms of estradiol secretion over negative control (agonist mode only). IC50: Concentration of compound that gave half-maximal inhibition of FSH-induced estradiol secretion (for antagonist mode only).
Hsueh, A. J., Bicsak, T., Jia, X.-C., Dahl, K. D., Fauser, B. C. J. M., Galway, A. B., Czwkala, N., Pavlou, S., Pakoff, H., Keene, J., Boime, I, xe2x80x9cGranulosa Cells as Hormone Targets: The role of Biologically Active Follicle-Stimulating Hormone in Reproductionxe2x80x9d Rec. Prog. Horm. Res., 1989, 45, 209-277.
The results obtained in this standard pharmacological test procedure are provided below.
Immature Rat Test Procedure
In order to assess the ability of compounds to affect FSH-induced changes in ovarian follicular maturation in vivo, immature rats (18 days of age) are treated twice daily for three days with compound in the presence of a half-maximal dose of purified human FSH. Animals are treated via the i.p. or p.o. routes. On the fourth day following treatment, animals are euthanized and the ovaries, uterus and spleen (the spleen is a control tissue that should not respond to FHS stimulation) collected for wet weight determination. In each experiment animals treated with vehicle alone or FSH alone are used as negative and positive controls, respectively. In this paradigm FSH induces a 2-3-fold increase in ovarian and uterine wet weight but has no effect on the spleen (control tissue).
The results obtained in this standard pharmacological test procedure are provided below.
Based on the results obtained in the standard pharmacological test procedures, the compounds of this invention were shown to antagonize the binding of hFSH to its receptor, in vitro, and to block cellular functions of FSH, in vitro, including the production of second messenger cAMP and estradiol in ovarian and granulosa cells. Representative compounds of this invention were also shown to inhibit FSH stimulated ovarian and uterine weight gain in immature female rats and ovulation in mature female rats. As such, the compounds of this invention are useful as female and male contraceptive agents.
The compounds of this invention may be administered orally or parenterally, neat or in combination with conventional pharmaceutical carriers. Applicable solid carriers can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintergrating agents or an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Liquid carriers may be used in preparing solutions, suspensions, emulsions, syrups and elixirs. The active ingredient of this invention can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fat. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (particularly containing additives as above e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used in sterile liquid form compositions for parenteral administration. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. Oral administration may be either liquid or solid composition form.
Preferably the pharmaceutical composition is in unit dosage form, e.g. as tablets or capsules. In such form, the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged compositions, for example packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form.
The therapeutically effective dosage to be used in the treatment of a specific psychosis must be subjectively determined by the attending physician. The variables involved include the specific psychosis or state of anxiety and the size, age and response pattern of the patient. In therapeutic treatment, projected daily dosages of the compounds of this invention are 0.1-500 mg/kg for oral administration.
The following procedures describe the preparation of representative examples of compounds of this invention.